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The m6A reader IGF2BP2 promotes pancreatic cancer progression through the m6A-SLC1A5-mTORC1 axis

Cancer Cell International volume 25, Article number: 122 (2025) Cite this article

Abstract

Background

Pancreatic cancer is a highly malignant digestive tumor. Glutamine metabolism is one of the important sources of tumors. N6-methyladenosine (m6A) modification plays a key role in regulating tumor metabolism and holds promise as a therapeutic target in various cancers, including pancreatic cancer. Disrupting m6A regulation of glutamine metabolism could impair tumor growth, offering potential new therapeutic strategies. However, the functional role of m6A modifications in pancreatic cancer, especially in glutamine metabolism, remains poorly understood.

Methods

The Cancer Genome Atlas (TCGA) dataset and GEPIA bioinformatics tool were used to identify the relationship between m6A related proteins and the glutamine metabolism-associated genes, respectively. The biological effects of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) were investigated using in vitro and in vivo models. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), MeRIP-PCR and RNA immunoprecipitation (RIP) were used to identify solute carrier family 1 member 5 (SLC1A5) as a direct target of IGF2BP2.

Results

We found that IGF2BP2 expression and SLC1A5 were significantly correlated and both highly expressed in pancreatic cancer could predict poor prognosis in patients with pancreatic cancer. Functionally, silencing IGF2BP2 suppressed tumor growth and also inhibited glutamine uptake by tumor cells. Mechanistically, IGF2BP2 induced the m6A-SLC1A5-mTORC1 axis, facilitating the uptake of glutamine by pancreatic cancer cells and accelerate the progress of pancreatic cancer. Furthermore, silencing IGF2BP2 can enhance the sensitivity of pancreatic cancer to radiotherapy and chemotherapy.

Conclusion

Our findings suggest that IGF2BP2 promotes pancreatic cancer by activating the m6A-SLC1A5 -mTORC1 axis. Targeting the m6A machinery, particularly IGF2BP2, offers a novel therapeutic avenue for pancreatic cancer treatment. By disrupting the regulation of glutamine metabolism, we provide new insights into how m6A-based therapies could enhance the efficacy of current treatments and offer hope for improving patient outcomes in this difficult-to-treat cancer.

Introduction

Pancreatic cancer, a highly malignant tumor of the digestive system, is the third leading cause of cancer deaths in both men and women, and the incidence and mortality of pancreatic cancer is increasing yearly. According to a statistical analysis of cancer deaths in the United States in 2024, pancreatic cancer has the lowest 5-year survival rate at 13% and remains a major global public health problem [1]. As pancreatic cancer patients usually have no symptoms in the early stage, making it difficult to detect the tumor, and pancreatic cancer has the characteristics of metastasis, recurrence, radiation resistance and chemotherapy resistance, making it lack of effective treatments, which also leads to the poor prognosis of pancreatic cancer patients in general and the overall survival rate is low, with the 5-year survival rate of only 12% [2, 3]. Hence, new therapeutic strategies are required to address these limitations. Genetic and epigenetic mutations are common in pancreatic cancer and are associated with aberrant activation of tumor driver genes [4]. However, the oncogenes involved in the development of pancreatic cancer tumors and their mechanisms of action are still ambiguous, there is an urgent need to explore them vigorously to improve the therapeutic efficacy of pancreatic cancer.

As a hallmark of tumors, metabolic reprogramming not only provides a source of energy for cancer cell growth, but also influences radiation therapy, chemotherapy, and immunotherapy for pancreatic cancer [5]. In addition to the Warburg effect and lipid metabolism, another common alteration in tumor metabolism is increased glutamine metabolism. Glutamine metabolism has a positive regulatory effect on the tricarboxylic acid cycle, fatty acid and nucleotide biosynthesis in tumor cells [6]. Importantly, compared to healthy individuals, inter-organ glutamine trafficking is significantly altered in tumor patients [7]. Decreased utilization and increased release of glutamine by organs, at the same time, tumor cells use glutamine to accelerate proliferation and growth, in addition glutamine can also affect tumor chemoresistance and resistance to radiotherapy [8,9,10,11]. Thus, glutamine metabolism may play an important role in pancreatic cancer growth and treatment. However, the mechanism of glutamine metabolism in promoting pancreatic cancer growth and influencing pancreatic cancer therapy remains unclear; therefore, we sought to explore the effects of glutamine metabolism on pancreatic cancer growth and therapy.

Methylation modification resulting in N6-methyladenosine (m6A) is the most prevalent internal modification, which can be involved in mechanisms related to pancreatic cancer development, progression, and immunoregulation, and the entire process of its functioning is involved by a series of proteins, which the researchers categorized into three groups, namely, methyltransferases, demethylases, and m6A-binding proteins, which are also imaginatively referred to as Writers, Erasers, and Readers [12,13,14,15,16]. In recent years, a large number of studies have demonstrated that m6A modifications can affect pancreatic cancer progression, participate in tumor metabolism, as well as alter the tumor microenvironment, which in turn affects the therapeutic efficacy of pancreatic cancer [17,18,19]. For example, METTL3 can promote pancreatic cancer progression and gemcitabine resistance by modifying DEAD-box helicase 23 (DDX23) mRNA m6A methylation and enhancing PI3K/Akt signaling activation [20].

IGF2BP2, a member of the insulin-like growth factor 2 messenger RNA-binding proteins (IGF2BPs) family, was initially identified as an RNA-binding protein that binds to IGF2 messenger RNA, and has since been shown to bind to a wide variety of other RNA transcripts, as well as to long-stranded, non-coding RNAs that are modified with N6-methyladenosine [21]. As an important recognition protein for m6A, IGF2BP2 can enhance mRNA stabilization and translation of oncogenes by recognizing m6A modifications, thereby promoting tumor progression [22]. The m6A modification affects mRNA stability and translation efficiency through its “read” proteins (e.g., IGF2BP2, etc.), thus promoting cancer cell growth and malignant transformation. A number of lines of evidence suggest that IGF2BP2 is a tumor promoter that can promote tumor growth and metastasis and participate in metabolism [22, 23]. However, little is known about the function and therapeutic potential of the IGF2BP2 protein, especially its m6A modification in pancreatic cancer, and further studies are warranted.

Herein, we report that IGF2BP2 can regulate glutamine metabolism through m6A modification, thereby affecting pancreatic cancer progression and treatment. First, we analyzed the relationship between m6A-related proteins and glutamine metabolism-associated genes by bioinformatics, and the results showed that the m6A-binding protein IGF2BP2 was closely related to the glutamine metabolism-associated genes Solute carrier family 1 member 5 (SLC1A5) in pancreatic cancer, and both IGF2BP2 and SLC1A5 were highly expressed in pancreatic cancer. Then, we experimentally demonstrated that IGF2BP2 regulates SLC1A5 expression in an m6A-modified manner, thereby affecting pancreatic cancer glutamine metabolism and decreasing the sensitivity of pancreatic cancer cells to radiation and chemotherapy, ultimately promoting pancreatic cancer progression. This study provides a potential prognostic biomarker and therapeutic target for pancreatic cancer patients.

Materials and methods

Tissue specimen collection and cell culture

All specimens were histopathologically confirmed by the pathologists. Patients or their relatives provided informed consent for use of tissue samples and data. This study was approved by the Ethics Committee of Jiangsu University.

The human pancreatic cancer cell lines PANC-1, MIA PaCa-2, AsPC-1, CFPAC-1 and SW1990 were purchased from the cell bank of the Typical Cultures Preservation Centre of the Chinese Academy of Sciences (Shanghai, China), Patu8988, and HPNE were purchased from the American Tissue Culture Conservation Center (ATCC, Manassas, Va. Manassas, VA, USA). These cells are immortalized pancreatic cancer cell lines, and the number of passages does not exceed 40.

Lentiviral transduction

We established IGF2BP2-knockout PANC-1cells and PaTu 8988 cells using the CRISPR-Cas9 genome editing system and generated stable SLC1A5 knockdown PANC-1cells and PaTu 8988 cells by transfecting synthesized short hairpin RNA (shRNA) of humanSLC1A5. The CRISPR-Cas9 genome targeting IGF2BP2 and and the lentivirus targeting SLC1A5 were purchased from Shanghai Genechem Co. The transfected cells were then selected with 5 µg/mL puromycin for 2 weeks. The efficiency of IGF2BP2 knockout and SLC1A5 knockdown were confirmed by western blot and qRT-PCR assays. The knockout efficiency of IGF2BP2 can reach more than 90%, and the knockdown efficiency of SLC1A5 can reach more than 75%. The sequences of sgRNA and shRNA have been detailed in Supplementary Table 2.

Western blot

Extract total protein from pancreatic cancer cells and then were subsequently combined with a volume of 5 × loading buffer and boiled for 10 min. Proteins were fractionated by 10% SDS-PAGE, transferred onto PVDF membranes, blocked in 5% BSA in TBS/Tween-20, and then blotted with specific antibodies. Antibodies used were as follows: anti-IGF2BP2 (ab124930, Abcam, UK) anti-SLC1A5 (ab237704, Abcam, UK), anti-mTOR (ab134903, Abcam, UK), anti-p-mTOR (ab109268, Abcam, UK), anti-P-P70 S6K (#9234, Cell Signaling Technology, USA), anti- P-eIF4EBP1 (ab278686, Abcam, UK), and GAPDH (YM3029, immunoway, USA). Secondary antibodies were obtained from immunoway.

Quantitative real-time PCR (qRT-PCR)

mRNA reverse transcription was performed using the HiScript® III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, R323-01, China), according to the manufacturer’s instructions. qRT-PCR was performed using the ChamQ SYBR qPCR Master Mix (Vazyme, Q311, China) with β-actin as the internal control for gene expression. The primer sequences used in this study are listed in Supplementary Table 2.

Cell proliferation assays

The cell counting kit-8 (CCK-8) (C0039, Beyotime, China) was used to evaluate cell proliferation according to the manufacturers’ instructions.

Colony formation assay

Eight hundred cells per well were seeded into 6-well plates and cultured in an incubator. Colonies comprising ≥ 50 cells were stained with 0.1% crystal violet dye subsequently counted and photographed to quantify the colonies formed.

Animal studies

Female nude BALB/c mice (6–8 weeks old) were used. For the subcutaneous transplantation model, 100 µL of 2 × 106 PANC-1 cells with or without KO-IGF2BP2 were injected subcutaneously into the right armpit of BALB/c nude mice at 6–8 weeks old. In addition, considering the effect of anesthesia on tumor growth, we used sodium pentobarbital to anesthetize nude mice [24]. Before radiotherapy, each mouse was intraperitoneally injected with sodium pentobarbital at a dose of 50 mg/kg. Tumor diameter was measured once a week from the time of implantation. The length (L) and width (W) of the tumor were monitored every 7 days with a vernier caliper. The tumor volume was calculated as follows: volume (mm3) = (W2 × L)/2.

Immunohistochemistry (IHC) staining and scoring analyses

Pancreatic cancer tissues were collected from the pathology department and fixed and embedded. After the gradient dewaxing treatment was completed, the dewaxed sections were subjected to antigen repair using antigen repair solution. The antibody (IGF2BP2: ab124930, Abcam; SLC1A5: 20350-1-AP, proteintech, China) was diluted with 1×PBS, and the diluted working solution was added dropwise to the tissues, placed in a wet box, and incubated at 4 °C overnight. On the next day, the sections were taken out, the antibody working solution was recovered, and washed with PBS for 3 times. After cleaned, diluted HRP-labeled goat anti-mouse IgG was added dropwise at a dilution ratio of 1:200, and incubated at 37 °C for 1 h. After DAB color development, the nuclei of the cells were protected from light, stained with hematoxylin dye, and then dehydrated with a gradient of ethanol in a range of concentrations from low to high, and then transparently processed in xylene, and finally incubated in a neutral resin. dendrimer sealing, after which the cells could be observed under an optical microscope. Immunohistochemical scoring criteria: place the immunohistochemical slides under a 100x microscope for observation, randomly select 5 fields of view for photography and recording, and score them according to the expression intensity and the percentage of positive staining area to the total area, 0 (no signal), 1 (weak), 2 (moderate), and 3 (strong). The staining distribution scores were determined according to the percentage of positive cells: 0 (0–5%), 1 (5–25%), 2 (25–50%), 3 (50–75%), 4 (75–100%). The median value was chosen as the cutoff.

mRNA stability assay

PANC-1 and PaTu8988 cells were inoculated in six-well plates, and the cells were treated with actinomycin D (5 µg/ml, GC16866, GLPBIO, USA) after 24 h. Subsequently, total RNA was extracted at 0 h, 3 h and 6 h, and then analyzed by qRT-PCR.

Methylated RNA Immunoprecipitation (MeRIP) assays

We used the ribo MeRIP m6A Transcriptome Profiling Kit (C11051-1, RiboBio, China) and performed MeRIP assays according to the manufacturer’s instruction. Firstly, we fragmented the total 100ug RNA into 100–150 bp fragments and used 1/10 of these fragments as input. Next, the remainder were conferred with anti-m6A antibody at 4 °C for 2 h. Finally, the m6A-modified mRNA was eluted from the magnetic beads with elution buffer and purified using Magen Hipure Serum/plasma miRNA Kit (R4317-03, Magen, China). The primer sequences used in this study are listed in Supplementary Table 2.

RNA Immunoprecipitation (RIP) assays

RNA Immunoprecipitation Kit (Bes5101, BersinBio, China) was used according to the manufacturer’s recommendation. The antibody used in this study is anti-IGF2BP2 (ab128175, Abcam, UK).

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software, USA). We applied two-tailed Student’s t-tests for comparisons of results between two different groups and one-way ANOVA or two-way ANOVA for multiple comparisons. Each experiment had a minimum of three replications. Statistical significance was determined as values of P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not statistically significant.

Results

Expression of IGF2BP2 is positively correlated with glutamine metabolism-associated gene SLC1A5 in pancreatic cancer and predicts poor prognosis of pancreatic cancer patients

Reprogrammed energy metabolism is an important feature of tumors, and one common metabolic change in tumors is an increase in glutamine metabolism. Glutamine, as the most abundant circulating amino acid in blood, plays a unique nutritional role in the growth and proliferation of tumor cells [9, 25, 26]. First, to define the level of glutamine metabolism in pancreatic cancer, we acquired a list of 41 glutamine metabolism-associated genes (Supplementary Table 1), and computed a score for the glutamine metabolism signature (Gln signature) by calculating the levels of The Cancer Genome Atlas (TCGA) PDAC database. The tumors were classified as Gln signature-high or Gln signature-low, and survival analysis showed that the Gln signature was significantly associated with poor OS (Fig. 1A, Supplementary Fig. 1A).

It was further illustrated by the ROC curve and area under the curve (AUC at 3 years = 0.690, AUC at 5 years = 0.910) that the score is a good predictor of prognosis in pancreatic cancer patients (Fig. 1B). These results suggested that compared to pancreatic cancer patients with less active tumor glutamine metabolism, patients with highly active tumor glutamine metabolism had poorer survival.

RNA m6A modification is essential for tumor metabolism. Then, we tried to investigate whether the pancreatic cancer intrinsic glutamine metabolism has relationship between m6A modification-associated genes (Supplementary Table 1). We conducted a correlation analysis between m6A modified genes and score, and found that IGF2BP2 had the most significant correlation (Supplementary Fig. 1B). Further analysis revealed that IGF2BP2 is closely related to the glutamine metabolism-associated genes SLC1A5 (Fig. 1C). Data mining of GEPIA database also showed a positive correlation between expression of IGF2BP2 and SLC1A5 (Fig. 1D), further providing evidence that IGF2BP2 is closely related to SLC1A5. These results supported the notion that IGF2BP2 may be involved in mediating the uptake of glutamine by tumor cells.

We employed bioinformatics-based methods to investigate the role of IGF2BP2 and SLC1A5 in human pancreatic cancer. The expression of IGF2BP2 and SLC1A5 were upregulated in pancreatic cancer tissues compared with that in normal tissues based on the GEPIA bioinformatics tool (Fig. 1E). Then, both GEPIA database and the bioinformatics tool Kaplan-Meier Plotter showed that patients with high IGF2BP2 or SLC1A5 expression had poorer overall survival (OS) than those with low IGF2BP2 or SLC1A5 expression (Fig. 1F, Supplementary Fig. 1C). Moreover, the bioinformatics tool GEPIA validated patients with pancreatic cancer exhibiting increased IGF2BP2 or SLC1A5 mRNA levels had worse DFS (Supplementary Fig. 1D).

Then, in order to further uncover the upregulation of IGF2BP2 and SLC1A5 expression in pancreatic cancer, we examined the expression of IGF2BP2 and SLC1A5 in human pancreatic cancer tissues. Consistent with the above research results, specific immunoreactivity of IGF2BP2 and SLC1A5 observed during malignant transformation and was found significantly increased in from PanIN lesions to pancreatic cancer (Fig. 1G and H). In addition, the correlation analysis of the immunohistochemical scores reconfirmed the positive correlation between expression of IGF2BP2 and SLC1A5 in pancreatic cancer tissues (Fig. 1I). These observations were further confirmed in the widely available KPC model. Compared with normal pancreatic ductal cells, IGF2BP2 and SLC1A5 are increased in cancerous ductal cells (Fig. 1J). Meanwhile, the expression of IGF2BP2 and SLC1A5 was commonly increased in pancreatic cancer cell lines compared with the nonmalignant hTERT-HPNE (hereinafter referred to as HPNE) cells (Fig. 1K, Supplementary Fig. 1E). In addition, CCK8 experiments further showed that glutamine deficiency significantly inhibited the growth of pancreatic cancer cells (Fig. 1L). Collectively, these findings indicate that IGF2BP2 was closely correlated with the expression of glutamine metabolism gene SLC1A5, which is up-regulated in pancreatic cancer, and its upregulation predicted poor prognosis of patients with pancreatic cancer.

Fig. 1
figure 1

IGF2BP2 is positively correlated with SLC1A5 and is associated with poor prognosis of pancreatic cancer. A. Kaplan-Meier survival analysis based on glutamine score in TCGA-PDAC cohort (P < 0.0001, log-rank test). B. ROC curve and AUC of glutamine level in patients with pancreatic cancer. (ROC = receiver operator characteristic. AUC = area under curve). C. Correlation between mRNA expression of IGF2BP2 and glutamine metabolism-associated genes in the TCGA database. D. Correlation between mRNA expression of IGF2BP2 and SLC1A5 in the GEPIA database. E. GEPIA database showing that the expression of IGF2BP2 and SLC1A5 in PANCREATIC CANCER tissues (T) and normal pancreas tissues (N). F. GEPIA database showing that overall survival of pancreatic cancer patients with diverse IGF2BP2 and SLC1A5 expression. G. Immunohistochemistry staining of IGF2BP2 and SLC1A5 in human pancreatic cancer. Scale bar, 2000 μm 20 μm. H. Immunohistochemical score statistical analysis. I. Correlation between the expression of IGF2BP2 and SLC1A5 based on immune score. J. Immunohistochemistry staining for KPC mice pancreatic tissue. Scale bar, 50 μm. K. The protein expression of IGF2BP2 and SLC1A5 were measured by western blot in the nonmalignant hTERT-HPNE (HPNE) cells and pancreatic cancer cell lines (PaTu8988, Mia-PaCa2, SW1990, PANC-1, CFPAC, Aspc-1). L. Viability of HPNE cells, PANC-1 cells and PaTu 8988 cells with or without glutamine analyzed by the CCK8 assay. *P < 0.05, ****P < 0.0001

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IGF2BP2 promotes tumor proliferation in vitro and in vivo by regulating glutamine metabolism

To evaluate the biological role of IGF2BP2 in pancreatic cancer, we generated IGF2BP2-knockout (KO) cells by utilizing the CRISPR-Cas9 system and determined the protein levels of IGF2BP2 in IGF2BP2-KO PANC-1cells and IGF2BP2-KO PaTu 8988 cells (Fig. 2A, Supplementary Fig. 2A). As shown by CCK8 and Colony formation tests, IGF2BP2 knockout remarkably suppressed the proliferation and colony formation of PANC-1 and PaTu 8988 cells (Fig. 2B and C). To further validate whether IGF2BP2 promoted tumor progression in vivo, we subcutaneously injected PANC-1cells into nude mice. The growth of IGF2BP2-KO transfected subcutaneous tumors was hindered compared to that of the controls (PANC-1 cells) (Fig. 2D). Meanwhile, the average tumor volume and weight at sacrifice were markedly decreased in mice with IGF2BP2-KO compared with the control mice (Fig. 2E and F). Further, significantly decreased proliferation was observed for the IGF2BP2-KO transfected subcutaneous tumors, as evidenced by the fact that Panc-1-IGF2BP2-KO cell-derived xenografts exhibited lower levels of Ki67 proteins (Fig. 2G). We further found by CCK8 and ELISA assay that deletion of IGF2BP2 leads to a decrease in the level of glutamine uptake by pancreatic cancer cells (Fig. 2H and I), which affects the proliferative capacity of tumor cells. These observations suggest that IGF2BP2 promotes the growth of pancreatic cancer in vitro and in vivo by regulating glutamine metabolism.

Fig. 2
figure 2

IGF2BP2 promotes proliferation and glutamine uptake in pancreatic cancer. A. Protein levels of IGF2BP2 in pancreatic cancer cell lines determined by western blot. B. Viability of PANC-1 cells and PaTu 8988 cells with or without IGF2BP2 knockout analyzed by the CCK8 assay. C. Representative images from the colony-forming assay and colony number analysis as indicated. D. Representative xenograft tumors after subcutaneous injection of PANC-1 cells transfected with KO-IGF2BP2 and KO-NC 42 days after inoculation. E. Tumor volume (n = 5) was recorded every week. F. Tumor weight (n = 5) of PANC-1 cells xenografts. G. Proliferation (Ki67) immunohistochemistry (IHC) staining of tumor sections. Scale bar, 50 μm. H. Viability of PANC-1 cells and PaTu 8988 cells with or without IGF2BP2 knockout analyzed by the CCK8 assay in the presence and absence of glutamine. I. Detection of intracellular glutamine levels through ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not statistically significant

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IGF2BP2 maintains SLC1A5 mRNA stability in an m6A-dependent manner

Recently, IGF2BP2 functions as an m6A reader, affecting mRNAs stability and translation of m6A-modified mRNAs [27]. To validate SLC1A5 as a potential target of IGF2BP2, IGF2BP2 was knocked out in pancreatic cancer cells, and our results showed that both protein and mRNA expression levels of SLC1A5 significantly inhibited by IGF2BP2 knockout (Fig. 3A and B, Supplementary Fig. 2B). Consistent with the decrease of protein and mRNA level, a shortening of mRNA half-life of SLC1A5 was observed upon knockout of IGF2BP2 (Fig. 3C).

To comprehensively profile genes with m6A modification mediated by IGF2BP2, we performed MeRIP-seq in PANC-1 cells with or without IGF2BP2 knockout. MeRIP-seq showed that PANC-1 cells had 11,499 unique peaks, while knockout of IGF2BP2 had 686 unique peaks (Fig. 3D). In addition, by analyzing the sequencing data, we found that GGAC was the most enriched motif found in m6A peaks identified from both IGF2BP2-KO cells and control PANC-1 cells, consistent with previous reports [28] (Fig. 3E). Moreover, from our MeRIP-seq data, we confirmed that m6A abundance in the SLC1A5 mRNA was diminished in response to IGF2BP2 knockout (Fig. 3F). These data together implicate an m6A-dependent regulatory mechanism.

Additionally, high-confidence m6A modification sites were predicted based on the m6A-seq data as well as the prediction from SRAMP (http://www.cuilab.cn/sramp) to determine the mechanism of m6A modification of SLC1A5 in pancreatic cancer (Fig. 3G), after which we designed primers at the very high-confidence m6A modification site (SLC1A5 # 1) and non m6A modification site (SLC1A5 # 2) respectively (Fig. 3H), and utilized Methylated RNA immunoprecipitation (MeRIP) to evaluate relative m6A abundance changes. MeRIP assays showed that upon knockout of IGF2BP2, the m6A abundance of SLC1A5 was significantly reduced at the modification sites, whereas the non-m6A modification sites were essentially unchanged (Fig. 3I). Moreover, the interaction between IGF2BP2 and SLC1A5 mRNA in both PANC-1 and PaTu 8988 cells was confirmed using the IGF2BP2-specifific antibody in RNA immunoprecipitation (RIP) assays (Fig. 3J). Collectively, these data establish that IGF2BP2 regulates SLC1A5 expression via m6A-dependent manner.

Fig. 3
figure 3

IGF2BP2 regulates SLC1A5 expression in an m6A-dependent manner. A. Protein levels of SLC1A5 in pancreatic cancer cell lines after IGF2BP2 knockout determined by western blot. B. mRNA levels of SLC1A5 in pancreatic cancer cell lines after IGF2BP2 knockout determined by RT-PCR. C. mRNA half-life (t1/2) of SLC1A5 in PANC-1 cells and PaTu 8988 cells with or without IGF2BP2 knockout. D. MeRIP-seq of PANC-1 cell lines showed a number of m6A peaks. E. MeRIP-seq analysis identified m6A motif. F. IGV visualization based on MeRIP-seq reveals significant m6A modification sites in the mRNA of SLC1A5. G. SRAMP online software predicted the m6A modification site of the mRNA of SLC1A5. H. Scheme showing the design of primers for MeRIP-qPCR to validate m6A modifications on SLC1A5 mRNA. Primer#1 is a potential m6A site, while primer#2 is a non m6A site. I. MeRIP-qPCR validated m6A modification with primers #1 and #2. J. The interaction between IGF2BP2 and the mRNA of SLC1A5 was detected by the RIP assay. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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SLC1A5 promotes pancreatic cancer proliferation by activating the mTORC1 signaling pathway

SLC1A5, an amino acid transporter, is frequently overexpressed in tumors and enhances glutamine metabolism [29]. Loss-of-function assays was used to assess the role of SLC1A5, a target gene of IGF2BP2, in pancreatic cancer. We knocked down the expression of SLC1A5 in pancreatic cancer cells and verified the knockdown efficiency by RT-PCR analysis (Fig. 4A). The CCK8 assay showed that depletion of SLC1A5 significantly suppressed the proliferation of pancreatic cancer cells compared to controls (Fig. 4B). Furthermore, the colony-forming ability of pancreatic cancer cells was remarkably inhibited when SLC1A5was knocked down (Fig. 4C). Together, the above data mimicked the effect of IGF2BP2 knockout.

In recent years, it has been found that amino acids are essential for mammalian target of rapamycin complex 1 (mTORC1) activation and that is particularly sensitive to decreases in glutamine [30,31,32], so we conjecture that SLC1A5 may promote the proliferation of pancreatic cancer cells by activating the mTORC1 signaling pathway. Therefore, we next dissected the role of SLC1A5 on mTORC1 signaling in pancreatic cancer. Our experimental results showed that knockdown of SLC1A5 inhibited mTORC1 pathway activation as evidenced by reduced phosphorylation of mTOR, ribosomal protein S6 kinase β-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) (Fig. 4D, Supplementary Fig. 2C). Additionally, the addition of glutamine reversed the results (Supplementary Fig. 3A). Furthermore, we found that knockdown of SLC1A5 may reduce glutamine uptake by pancreatic cancer cells (Fig. 4E). We next dissected the role of IGF2BP2 on mTORC1 signaling in and the results showed that knockout of IGF2BP2 inactivated the mTORC1 pathway (Supplementary Fig. 3B and D-E). Taken together, our data show that a unique molecular association between SLC1A5 and mTORC1 signaling in PANCREATIC CANCER.

Fig. 4
figure 4

SLC1A5 promotes pancreatic cancer cell proliferation and glutamine uptake through activation of the mTORC1 pathway. A. mRNA levels of SLC1A5 in PANC-1 and PaTu 8988 cells determined by RT-PCR. B. Viability of PANC-1 cells and PaTu 8988 cells with or without SLC1A5 knockdown analyzed by the CCK8 assay. C. Representative images from the colony-forming assay and colony number analysis as indicated. D. Depletion of SLC1A5 inactivated mTORC1 signaling by western blot. E. Detection of intracellular glutamine levels through ELISA. **P < 0.01, ***P < 0.001, ****P < 0.0001

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IGF2BP2 is a promising therapeutic target in pancreatic cancer

The above data together demonstrated that IGF2BP2, through regulating glutamine metabolism pathways, is critical for promoting the growth of pancreatic cancer cells. Considering the critical role of glutamine cleavage in pancreatic cancer, we tested whether IGF2BP2 could be an attractive target for pancreatic cancer therapy.

To investigate the effect of IGF2BP2 on cell radiosensitization, we performed CCK8 experiments and colony formation experiments, which showed that although KO-IGF2BP2 or 6GY irradiation individually inhibited cell proliferation, their combination showed a more significant inhibitory effect compared to either KO-IGF2BP2 or 6GY irradiation alone (Fig. 5A and B). Additionally, we established a xenograft mouse model to confirm that IGF2BP2 has the same effect in vivo. In accordance with the cell-based results, IGF2BP2-KO cells treated with 6GY irradiation markedly repressed tumor growth in mice, as reflected by the significant inhibition of the tumor volume and weight when compared to the control group (Fig. 5C and D).

Currently, gemcitabine is still the first-line drug for the treatment of pancreatic cancer, but its chemotherapy resistance creates significant resistance to the treatment. Therefore, enhancing sensitivity to gemcitabine may contribute to enhancing the effects of therapy and improving the prognosis of pancreatic cancer. We hypothesized that the combination of knockout IGF2BP2 and gemcitabine might achieve a better effect in pancreatic cancer treatment. To this end, PANC-1 and PaTu 8988 cells with or without IGF2BP2 knockout were treated with gemcitabine. As expected, combination treatment resulted in more significant inhibition on the growth of pancreatic cancer cells (Fig. 5E).

To determine whether IGF2BP2 has a role in mediating gemcitabine sensitivity in pancreatic cancer cells in vivo, we established a subcutaneous xenograft tumor model with PANC-1 cells with or without KO-IGF2BP2, followed by intraperitoneal administration of gemcitabine 2 times a week. Targeting IGF2BP2 or administration of gemcitabine alone modestly reduced tumor size and weight as compared to control. Additionally, combination treatment resulted in more significant suppression of PANC-1 xenograft tumor growth (Fig. 5F and G). Our data collectively show that knockout IGF2BP2 can not only enhance the radiosensitivity of pancreatic cancer, but also enhance the chemosensitivity of pancreatic cancer, providing a new direction for improving the therapeutic effect of pancreatic cancer.

Fig. 5
figure 5

Knockout of IGF2BP2 enhances sensitivity of pancreatic cancer cells to radiotherapy and chemotherapy. A. Viability of PANC-1 cells and PaTu 8988 cells with or without IGF2BP2 knockout under 6 Gy radiation therapy and 0 Gy radiation therapy analyzed by the CCK8 assay. B. Representative images from the colony-forming assay and colony number analysis as indicated. C. The effects of 6 Gy radiation treatment on the growth of subcutaneous PANC-1 cells with or without IGF2BP2 knockout xenografts. D. Tumor weight (n = 5) of PANC-1 cells xenografts. E. PANC-1 and PaTu 8988 cells with or without IGF2BP2 knockout were treated with gemcitabine at different concentrations for 48 h, and cell viability was then measured by CCK8 assay. F. Effect of gemcitabine treatment on the growth of subcutaneous PANC-1 cells with or without IGF2BP2 knockout xenografts. G. Tumor weight (n = 5) of PANC-1 cells xenografts

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Discussion

In recent years, it has been found that reprogramming of glutamine metabolism contributes to the proliferation of tumor cells and enhances their viability, in addition to affecting the sensitivity of tumor cells to radiotherapy and chemotherapy [33, 34]. Notably, m6A-related proteins can regulate the expression of potential tumor targets in an m6A-dependent manner, which can alter tumor invasion and proliferation, regulate tumor metabolism, and influence tumor treatment outcomes [35,36,37]. In this study, we report that m6A modification plays a key role in the regulation of glutamine metabolism in pancreatic cancer, in which IGF2BP2 mediates the effect of m6A modification on the expression of the critical gene SLC1A5, allowing for the rapid proliferation and survival of pancreatic cancer cells. In addition, we have elucidated that SLC1A5-mTORC1 is a key target of IGF2BP2 in pancreatic cancer, and that targeting IGF2BP2 expression level affects the sensitivity of radiation and chemotherapy for pancreatic cancer, suggesting that IGF2BP2 is a potential key target for improving the efficacy of pancreatic cancer treatment.

Glutamine metabolism is the key to tumor cell growth and proliferation. Glutamine, as a non-essential amino acid, on the one hand, provides a ready source of carbon and nitrogen to meet the metabolic needs of tumor cell growth; on the other hand, it reduces the sensitivity of tumors to chemotherapy [38,39,40]. It is noteworthy that the two pathways, glutamine metabolism and Warburg effect, work together in cancer cells to satisfy the energy and biosynthetic intermediates required for rapid proliferation of cancer cells.

Additionally, because the Warburg effect leads to a decrease in pyruvate entering the TCA cycle, cancer cells rely on intermediates such as α-ketoglutarate produced by glutamine metabolism to replenish the TCA cycle, thereby maintaining their energy metabolism and biosynthesis processes, indicating that glutamine metabolism provides tumor cells with an additional source of energy, supplementing the energy requirements of cells under the Warburg effect. Activation of mTORC1 downstream of glutamine uptake further facilitates the metabolic reprogramming characteristic of cancer cells. This includes increased glycolysis and lactate production, hallmark features of the Warburg effect, even in the presence of oxygen [41]. By driving the expression of glycolytic enzymes and inhibiting autophagy, mTORC1 shifts the metabolic balance toward biomass accumulation and rapid proliferation, processes essential for tumor progression. In this study, we found that IGF2BP2 provides energy for pancreatic cancer cell growth through activation of the m6A-SLC1A5-mTORC1 axis, compensating for the reduction in the oxidative phosphorylation pathway in cancer cells caused by the Warburg effect.

we found that glutamine metabolism levels were strongly associated with overall survival in pancreatic cancer through bioinformatics analysis, as reflected by the higher the glutamine metabolism levels, the lower the overall survival of pancreatic cancer patients. Considering the critical role of m6A modification in glutamine metabolism [40], we went on to identify a positive regulatory relationship between the m6A-binding protein, IGF2BP2, and the key gene for glutamine metabolism, SLC1A5, through bioinformatics analysis. When using the TCGA database, we also considered the problem of data bias and tried to reduce and correct the bias as much as possible [42]. Further studies revealed that both IGF2BP2 and SLC1A5 were up-regulated in pancreatic cancer and were strongly associated with its poor prognosis. Indicating that IGF2BP2 and SLC1A5 may be important oncogenes.

As an m6A-bingding protein (reader), IGF2BP2 acts as an m6A reader to stabilize and promote translation of mRNAs that encode proteins involved in tumor progression [43]. IGF2BP2 has been implicated in various cancer types. In hepatocellular carcinoma (HCC), IGF2BP2 can stabilize CDC45 mRNA through m6A modification, thereby promoting HCC glycolysis and stemness [44]. Furthermore, IGF2BP2 can promote lymphatic metastasis of papillary thyroid cancer by stabilizing DPP4 in an m6A-dependent manner [45]. These studies highlight the role of IGF2BP2 in promoting tumor progression by stabilizing oncogenic transcripts in different cancers. IGF2BP2 is specifically upregulated in pancreatic cancer, whereas its expression is low in normal pancreatic tissue, making it a potential target for pancreatic cancer therapy. In pancreatic cancer, IGF2BP2 can promote aerobic glycolysis and pancreatic cancer cell proliferation by stabilizing glucose transporter protein 1 (GLUT1) mRNA [46]. In the tumor microenvironment, SLC1A5 plays an important role in inducing glutamine uptake by tumor cells as a major transporter protein for glutamine uptake [26]. It was found that SLC1A5 expression was elevated in a variety of tumors (triple-negative breast cancer, melanoma, and prostate cancer), and blockade of SLC1A5 inhibited the uptake of glutamine by tumor cells, thereby inhibiting tumor cell growth [29, 47, 48]. However, the mechanism of action by which IGF2BP2 regulates SLC1A5 to mediate glutamine metabolism in pancreatic cancer is unclear. Our data suggest a unique mechanism in pancreatic cancer in which IGF2BP2 acts as an m 6 A reader to stabilize and facilitate translation of mRNA encoding SLC1A5, a protein involved in glutamine metabolism. Whether this m6A mechanism exists in other solid tumors remains to be explored.

The mTORC1 signaling pathway not only regulates tumor cell growth and protein translation, but also plays an important role in cellular uptake of glutamine [49]. In addition, targeting inhibition of the mTORC1 signaling pathway by rapamycin has been considered as a potential approach for cancer therapy. Notably, both glutamine metabolism and the Warburg effect are involved in the mTOR signaling pathway, which together regulate biological behaviors such as growth and proliferation of tumour cells. To this end, we experimentally identified SLC1A5-mTORC1 as the primary target of IGF2BP2 in pancreatic cancer.

Owing to the highly fibrotic nature of pancreatic cancer, drug delivery is compromised, and therapy resistance [50, 51]. Despite gemcitabine being a first-line agent for the treatment of pancreatic cancer, especially in patients with advanced pancreatic cancer that has released metastases, it significantly inhibits therapeutic efficacy due to its drug resistance [52]. Currently, chemoresistance and radioresistance are one of the important reasons for the poorer prognosis of pancreatic cancer; therefore, enhancing the sensitivity of pancreatic cancer to radiotherapy and chemotherapy is crucial for improving the prognosis of pancreatic cancer patients. The current study found that IGF2BP2 mainly promotes tumor proliferation and metastasis [53], but there are no studies of IGF2BP2 resistance to chemotherapy and radiotherapy. Remarkably, our study found that IGF2BP2 depletion in pancreatic cancer could enhance its sensitivity to radiotherapy and chemotherapy, which brings new hope for the treatment of pancreatic cancer. On the one hand, by inhibiting the expression or function of IGF2BP2, the SLC1A5-mTORC1 cancer signaling pathway can be blocked to inhibit cancer proliferation, and on the other hand, targeting IGF2BP2 may synergize with other therapeutic approaches (e.g., chemotherapy, radiotherapy) to improve therapeutic effects. A large number of studies have shown that local anesthetics have a certain effect on the proliferation and metastasis of cancer cells, so we finally chose sodium pentobarbital to anesthetize the nude mice [24].

In conclusion, our studies identified the vital role of IGF2BP2, as an m6A-binding protein in controlling glutamine metabolism during the pathogenesis of pancreatic cancer, and highlight that IGF2BP2 promotes tumor progression through the m6A-SLC1A5- mTORC1 axis. Targeting IGF2BP2 could disrupt these pathways, leading to reduced tumor progression. However, RNA modifications are ubiquitous, so targeting m6A mechanisms may affect normal cellular processes, requiring the development of highly selective inhibitors. In addition, the role of m6A modifications may vary by cancer type, leveraging single-cell RNA sequencing and spatial transcriptomics to tailor therapies to specific tumor conditions. In future work, we can try to test IGF2BP2 or SLC1A5 targeting strategies in pancreatic cancer patient-derived xenograft models and then validate them in larger multicenter patient cohorts to further explore the clinical value of IGF2BP2 and SLC1A5 as biomarkers or therapeutic targets. In addition, our future studies can explore whether external factors regulate the IGF2BP2-m6A-SLC1A5-mTORC1 axis [54].

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We sincerely thank to all team members for their assistance for this work.

Funding

This work was supported by the National Natural Science Foundation of China (grant no. 82072754), the Science and Technology project of Jiangsu Provincial Health Commission (grant no. M2020011), the Key R&D Programs of Jiangsu Province (grant no. BE2018689), the Key R&D Programs of Zhenjiang City (grant no. SH2018033), the National Natural Science Foundation of China (grant no. 32170910), the Natural Science Foundation of Jiangsu Province (grant no. BK20211124) and the Zhenjiang Key Research and Development Program (grant no. SH2021037), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX23_3757).

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Author notes

  1. Xi Pu, Yuting Wu, Weiguo Long and Xinyu Sun contributed equally to this work.

Authors and Affiliations

  1. Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, 212001, China

    Xi Pu, Yuting Wu & Min Xu

  2. Pathology Department, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, 212001, China

    Weiguo Long

  3. Department of Otorhinolaryngology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China

    Xinyu Sun

  4. Department of Radiation Oncology, Institute of Oncology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, 212001, China

    Xiao Yuan, Deqiang Wang & Xu Wang

  5. Institute of Digestive Diseases, Jiangsu University, Zhenjiang, Jiangsu, 212013, China

    Deqiang Wang & Min Xu

  6. Excellent Medical School, Jiangsu University, Zhenjiang, Jiangsu, 212013, China

    Min Xu

  7. No. 438 Jiefang Road, Jingkou District, Zhenjiang, Jiangsu Province, 212001, China

    Deqiang Wang, Xu Wang & Min Xu

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Contributions

XP, YTW and XYS performed the experiments and wrote the manuscript; WGL and XY analyzed the data; DQW, XW and MX supervised the study and edited the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Deqiang Wang, Xu Wang or Min Xu.

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The study was conducted in accordance with the Helsinki Declaration and approved by the Medical Ethics Committee of Jiangsu University for research involving humans. The animal research protocol has been approved by the Experimental Animal Care Management Committee of Jiangsu University (UJS-IACUC-AP-2020041402).

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Pu, X., Wu, Y., Long, W. et al. The m6A reader IGF2BP2 promotes pancreatic cancer progression through the m6A-SLC1A5-mTORC1 axis. Cancer Cell Int 25, 122 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03736-8

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Cancer Cell International

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